DEVELOPMENTAL DYNAMICS 218:112–122 (2000) Neural and Hormonal Control of Expression of Myogenic Regulatory Factor Genes During Regeneration of Xenopus Fast Muscles: Myogenin and MRF4 mRNA Accumulation Are Neurally Regulated Oppositely NATHALIE NICOLAS,1 JEAN-CLAUDE MIRA,2 CLAUDE L. GALLIEN,1 AND CHRISTOPHE CHANOINE1* 1 Laboratoire de Biologie du Développement et de la Différenciation Musculaire, Paris, France 2 Laboratoire de Neurobiologie; Université René Descartes, Paris, France ABSTRACT With the aim to investigate the influence of both innervation and thyroid hormone, on the expression of the MRFs during muscle regeneration, we performed cardiotoxin injury-induced regeneration experiments on fast muscles of adult Xenopus laevis subjected to different experimental conditions, including denervation and T3 treatment, and analyzed the accumulation of the four myogenic regulatory factors (MRFs) using RT-PCR and in situ hybridization. We show here that manipulation of hormone levels or innervation resulted in differential alterations of MRF expression. Denervation and T3 treatment transiently down-regulated Myf-5 mRNA levels at the beginning of the regeneration process. Myf-5 was the only myogenic factor subject to thyroid hormone influence. Muscle denervation persistently reduces the levels of MRF4 transcripts as early as the first stages of regeneration, whereas the levels of myogenin mRNA were increased in the late stages of regeneration. This suggests that MRF4 expression may be induced by innervation and hence may be involved in mediating transcriptional responses to innervation and that myogenin expression may compensate for the down-regulation of MRF4 gene. This switch in MRF gene expression following denervation could have important consequences for the ability of Xenopus regenerating muscles to recover function after denervation. Dev Dyn 2000;218:112–122. © 2000 Wiley-Liss, Inc. Key words: muscle regeneration; myogenic regulatory factors; innervation; thyroid hormone; Xenopus INTRODUCTION The four myogenic regulatory factors (MRFs): MyoD (Davis et al., 1987), Myf-5 (Braun et al., 1989), myogenin (Wright et al., 1989), and MRF4 (Rhodes and Konieczny, 1989; Braun et al., 1990; Miner and Wold, 1990), are basic helix-loop-helix transcription factors whose ectopic expression is able to convert a wide range of cultured cells to a muscle phenotype (Schäfer et al., 1990; Choi et al., 1990) and which can promote © 2000 WILEY-LISS, INC. the transcription of a number of muscle-specific genes (Weintraub et al., 1991). The functions of the MRFs in vivo have been investigated by determining the pattern of their expression, by gene targeting and ectopic expression. Gene knock-out mice experiments have elucidated the hierarchical relationships existing between the MRFs. MyoD and Myf-5 are required for the determination of skeletal myoblasts, and myogenin and MRF4, act later in the program, probably as differentiation factors (reviewed in Buckingham, 1994; Ludolph and Konieczny, 1995). During development, the order of expression of MRF genes varies according to muscle origin and between species. In Xenopus, the key difference lies in the observation that XMyoD and XMyf-5 mRNA can be detected in presomitic mesoderm (Hopwood et al., 1989; 1991), which is in contrast to the murine system. Moreover, myogenin transcript was only detected during secondary myogenesis of Xenopus and never during somitogenesis (Jennings, 1992; Nicolas et al., 1996, 1998a). An important feature of mature skeletal muscles is their ability to regenerate following injury. Satellite cells, closely associated with muscle fibers, are myoblast-like cells responsible for the regenerative capacity of muscles (Campion, 1984). These adult muscle stem cells are normally mitotically quiescent but are activated in response to injury. The regenerative process is characterized by the proliferation of the descendants of the activated satellite cells, called myogenic precursor cells (mpc), before fusing to form new myotubes which differentiate into mature myofibers. Given the importance of the MRFs for myoblast differentiation during development, the pattern of expression of the MRFs has been analyzed during muscle regeneration in mammals (Grounds et al., 1992; Füchtbauer et al., 1992; Kami et al., 1995; Rantanen et al., 1995) and amphibians (Nicolas et al., 1996, 1998b) following different types of injury. However, little is known about *Correspondence to: Pr. C. Chanoine, Laboratoire de Biologie du Développement et de la Différenciation Musculaire (EA 2507), Centre Universitaire des Saints-Péres, Université René Descartes, 45 rue des Saints-Péres, 75270 Paris cedex 06, France. E-mail: firstname.lastname@example.org Received 13 September 1999; Accepted 31 Janaury 2000 CONTROL OF EXPRESSION OF MYOGENIC REGULATORY FACTOR GENES 113 Fig. 2. RNAse protection for acetylcholine receptor (␣ subunit) and EF-1␣ RNA. Amounts used were 5 g total RNA per assay; innervated (C) and denervated (D) regenerating muscles taken at 15 days following cardiotoxin injury. Fig. 1. Analysis of serum thyroid hormone levels in control adult animal (C), T3 treated adult animal (T3) and prometamorphic larvae (M). the specific involvement of innervation and thyroid hormone in the accumulation of the MRF transcripts during muscle regeneration (Koishi et al., 1995). Yet, it is well known that these two factors have a critical role in determining adult muscle phenotype. It is also well established that embryonic myoblasts and satellite cells are not equivalent cells (Stockdale, 1992) which are submitted to distinct neural and hormonal environment. Moreover innervation and thyroid hormone are able to regulate MRF expression in vitro (reviewed in Muscat et al., 1995), and are also controlling factors in adult muscles (Hughes et al., 1993; Adams et al., 1995). Therefore, these epigenetic factors could be involved in MRF expression following muscle injury in adult animals. In this work, to investigate the influence of both innervation and thyroid hormone on the expression of the MRFs during muscle regeneration, we performed cardiotoxin injury-induced regeneration experiments on fast muscles of adult Xenopus laevis subjected to different experimental conditions, including denervation and T3 treatment, and analyzed the accumulation of MyoD, Myf-5, myogenin, and MRF4 using RT-PCR and in situ hybridization. We show here that manipulation of hormone levels or innervation resulted in alterations of MRF expression. These results are discussed in relation to the ability of Xenopus regenerating muscles to recover function following epigenetic changes. RESULTS The sequence of histological changes as well as the pattern of expression of MRFs observed in Xenopus regenerating muscle following cardiotoxin injury has been previously described (Saadi et al., 1994; Nicolas et al., 1996, 1998b). A single injection of cardiotoxin caused an almost complete degeneration of the myofi- bers within 24 hr. In the present study, regenerative stages corresponded to the following days: mononucleate cells/young myotubes, 11 days postinjection; young myotubes, 15 days postinjection; large myotubes, 20 days postinjection; and mature myofibers, 1 month postinjection. To be sure of the completeness of the degeneration/ regeneration process, all muscle samples were monitored as indicated in Experimental Procedures. To analyze the accumulation of MRF transcripts during muscle regeneration, we chose to use both in situ hybridization and a semi-quantitative RT-PCR assay able to detect small amounts of transcripts and thus offering the opportunity to analyze small samples (Nicolas et al., 1998a). The analyzed transcripts were co-reverse-transcripted and co-amplified in the same reaction with the ubiquitously expressed EF-1␣ gene (Krieg et al., 1989) to serve as internal control for the amount of RNA tested and for the RT-PCR reproducibility. Thyroid hormone is required for the normal development of the central nervous system (Dussault and Ruel, 1987) and probably peripheral nerves, which suggested that the regulation by thyroid hormone of some muscle-specific genes could be nerve-mediated. To determine whether T3 acts via the nerve innervating the brachial muscle, we removed a portion of one of the two brachial nerve in adult Xenopus just before cardiotoxin injection in the two anterior brachial muscles of the same animal. One set was treated with T3 and another set was untreated. The innervated contralateral muscles of the operated animals were compared to the denervated muscles since both should be exposed to the same circulating levels of T3, thus providing a better control for this parameter than independently T3 treated Xenopus. The hyperthyroidian status of the T3-treated animals was monitored by measuring the circulating T3 levels (Fig. 1). In denervated regenerating muscles, AChR ␣ subunit RNA was strongly induced, confirming that denervation was successful, while another control RNA, EF-1␣ (Krieg et al, 1989), was unaffected by denervation (Fig. 2 and Jennings, 1992). At the first stage of muscle regeneration analyzed (11 days P-I), semi-quantitative RT-PCR showed a strong decrease of Myf-5 mRNA levels as much as 3– 4-fold, following denervation as well as T3 treatment 114 NICOLAS ET AL. Fig. 3. Semi-quantitative analysis of MRF expression at different stages of regeneration (11 days, 15 days, 20 days, and 30 days P-I) and in different experimental conditions assayed by RT-PCR amplification. A: Myf-5, B: MyoD, C: MRF4, and D: myogenin. Intensity of each PCR product signals was quantified after scanning with the NIH Image analyzer software. The MRF signal was normalized to EF-1␣ signal, and the relative amount of each sample PCR product is presented. Five distinct RT-PCR analysis for one sample were performed. *significantly different from C value, P ⬍ 0.05. C, natural hypothyroidism; T3, T3 treatment; D, denervation; DT3, T3 treatment and denervation. (Fig. 3A). This down-regulation of Myf-5 gene by our experimental treatments was confirmed by in situ hybridization (Fig. 4). The fact that T3 treatment induced a similar effect on Myf-5 gene expression in innervated as well as in denervated regenerating muscles showed that T3 acts independently of innervation. This demonstrated that muscle denervation and T3 treatment were involved in the down-regulation of Myf-5 gene expression in two distinct ways. From day 15 P-I, both denervation and/or T3 treatment have no significant effects on the expression of Myf-5 (Fig. 3A). Semi-quantitative RT-PCR revealed that, at all stages of the regenerating process, neither denervation nor T3 treatment modified the MyoD mRNA levels (Fig. 3B). From 11 days P-I to 1 month P-I, RT-PCR analysis showed that MRF4 mRNA was significantly downregulated by denervation whereas T3 treatment had no effect on the accumulation of this MRF mRNA (Fig. 3C). In contrast to what was observed for MRF4, muscle denervation increases the levels of myogenin mRNA in the late stages of muscle regeneration, from 20 days P-I (Fig. 3D). These results were confirmed by in situ hybridization: as early as the first stage of regeneration (11 days P-I), the hybridization signal for MRF4 transcripts was significantly reduced in denervated muscles in comparison to that observed in innervated contralateral muscles as well as in innervated muscles of T3treated animals (Fig. 5). This fall of the level of MRF4 mRNA due to muscle denervation was continuously detected in the following stages of regeneration as shown in large myotubes at 20 days P-I (Fig. 6). In situ hybridization experiments also clearly confirmed that T3-treatment has no effect on the accumulation of MRF4 mRNAs during muscle regeneration (Fig. 5C and D; Fig. 6D and E). For myogenin transcripts, as early as the large myotube stage, the hybridization signal detected in denervated muscles of control as well as T3-treated animals was strong in comparison to innervated contralateral muscles where myogenin transcripts were not detected using in situ hybridization, in accordance with our previous results (Nicolas et al., 1996) (Fig. 7). It should be noted that the response of muscle to denervation is more belated in the case of myogenin in comparison to that of MRF4: the down-regulation of MRF4 is observed as early as the beginning of the regenerating process, whereas the up-regulation of the myogenin gene only appears in the late stages of muscle regeneration (Fig. 3 D). Like that observed for MRF4, the accumulation of myogenin transcripts is unaffected by T3 treatment (Figs. 3D, 7). DISCUSSION This article provides a detailed analysis of both the neural and the hormonal influences on the expression of the MRF genes during regeneration of Xenopus fast muscles. It shows that in Xenopus, the accumulation of each MRF mRNA is differentially regulated by innervation and/or thyroid hormone during in vivo myogenesis. Thus, each member of the MyoD family may have a distinct role in the maintenance of the specific physiological properties of Xenopus muscle. CONTROL OF EXPRESSION OF MYOGENIC REGULATORY FACTOR GENES 115 Fig. 4. In situ hybridization using antisense riboprobes to Myf-5 on transverse sections of control (A, D), T3-treated (B) and denervated (C) regenerating muscle at 11 days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). A, B and C are darkfield photomicrographs. D is a brightfield photomicrograph of the same section that is shown in A. Scale bar ⫽ 200 m Myf-5 Is the Only MRF Subject to Hormonal Regulation During Muscle Regeneration of Xenopus mRNA in slow twitch soleus fibers (Hughes et al., 1993). From these results, it has been hypothetized that, at the level of the muscle cells, one or several members of the MyoD family could mediate the effects of TH on some contractile protein genes (Muscat et al., 1995). In Xenopus, the correlative accumulation of myogenin and fast MHC mRNAs in secondary myofibers, when TH levels increase during metamorphosis, had also suggested this type of regulation (Nicolas et al., 1998a). Surprisingly, in the present study, we find that T3 treatment does not up-regulate any of the four MRF mRNA levels. In contrast, Myf-5 transcripts were down-regulated by T3 in the first step of the regeneration process. To our knowledge, this down-regulation of Myf-5 by TH has never been described in any in vitro or in vivo models and should be considered in relation to the potential involvement of Myf-5 at the beginning of myogenesis. It appears that during three types of myogenesis in Xenopus, including somitogenesis, regeneration and limb It is known that thyroid hormone is a major controlling influence of myogenesis. T3 regulates the expression of many muscle-specific genes (d’Albis et al., 1987; Chanoine et al., 1987, 1989; Saadi et al., 1993). It has been observed that T3 treatment of the myogenic cell line, C2.7, promoted terminal differentiation, increased MyoD gene transcription and resulted in the precocious expression of myogenin and contractile protein mRNAs (Carnac et al., 1992). Furthermore, it was observed that overexpression of the c-erbA ␤ gene encoding thyroid hormone receptor (TR) in myogenic cells enhanced differentiation and increased MyoD expression in the presence of T3 (Begue et al., 1993). In the rat, it has also been demonstrated that MyoD selectively accumulates in fast twitch fiber types and that thyroid hormone treatment results in the significant induction of MyoD and fast IIa MHC 116 NICOLAS ET AL. Fig. 5. In situ hybridization using antisense riboprobes to MRF4 on transverse sections of control (C), T3-treated (D) and denervated (A,B) regenerating muscle at 11 days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). B, C and D are darkfield photomicrographs. A is a brightfield photomicrograph of the same section that is shown in B. Scale bar ⫽ 200 m development (Hopwood et al., 1989, 1991; Rupp and Weintraub, 1991; Nicolas et al., 1996, 1998a and b), MyoD and Myf-5 are the first myogenic factors expressed. Microinjection of synthetic XMyf-5 or XMyoD mRNA into early embryos suggest that XMyf-5 might act largely during the very early stages of myogenesis before MyoD. It is also interesting to note that the XMyf-5 gene is only transiently expressed in all types of myogenesis in Xenopus whereas XMyoD is continuously expressed, XMyoD mRNA being the major MRF transcript detected in Xenopus adult muscle (Jennings, 1992). In tissue culture, the differentiation of myoblasts is tightly regulated through a suppression mechanism mediated by a gamut of oncogene products and growth factors which prevent cell cycle arrest and repress trans-activation of myogenic gene expression. Hormonal stimulation (thyroid hormone, retinoic acid (RA) and insulin-like growth factors (IGFs)) and growth factor deprivation induce proliferating myoblasts to exit the cell cycle and fuse into post-mitotic multinucleated myofibers that express muscle-specific phenotypic markers (rev. in Muscat et al., 1995). In particular, it has been observed that all-trans and 9-cis RA repress the Myf-5 gene at the transcriptional level (Carnac et al., 1993). We can suggest that the increase of circulating T3 levels in TH-treated animals forced the proliferating myoblasts to differentiate, involving the downregulation of Myf-5 gene and the expression to musclestructural genes. We have recently shown that T3 upregulated the transcripts coding for the fast MHC as early as the first stages of muscle regeneration (unpublished results). Myogenin and MRF4 mRNA Accumulation Are Neurally Regulated Oppositely It is known that innervation induces many changes in muscle gene expression (reviewed in Laufer and Changeux, 1989). Many of these are caused by electri- CONTROL OF EXPRESSION OF MYOGENIC REGULATORY FACTOR GENES 117 Fig. 6. In situ hybridization using antisense riboprobes to MRF4 on transverse sections of control (A, C), T3-treated (D), denervated (B), and denervated/T3-treated (E) regenerating muscle at 20 days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). B, C, D, and E are darkfield photomicrographs. A is a brightfield photomicrograph of the same section that is shown in C. A–C: Scale bar ⫽ 200 m; D, E: Scale bar ⫽ 150 m cal activity in the muscle, for example the suppression of acetylcholine receptor expression in extrajunctional regions of muscle fibers or the expression of particular MHC isoforms (Goldman et al., 1988; Kirschbaum et al., 1990). Many studies have shown that the loss of electrical stimulation can lead to up-regulation of myogenic HLH mRNAs (Eftimie et al., 1991; Piette et al., 1992; Hughes et al., 1993), suggesting that the myogenic HLH transcription factors could mediate the effects of the nerve. In mammals, Duclert et al. (1991) showed that MRF4 mRNA as well as myogenin mRNA were induced by denervation. From the literature, it seems that these two MRFs also play the central role in mediating nerve influence on muscle gene expression (Sunyer et Merlie, 1993; Buonanno et al., 1993; Merlie et al., 1994). Our study reveals that myogenin and MRF4 mRNAs are regulated in two opposite ways by denervation in contrast to what is observed in mammals (Adams et al., 1995). We show that muscle denervation persistently reduces the levels of MRF4 transcripts during regeneration whereas the levels of myogenin mRNA increase in the late stages of regeneration. Jennings (1992) has also previously shown that a short-term denervation 118 NICOLAS ET AL. Fig. 7. In situ hybridization using antisense riboprobes to myogenin on transverse sections of control (C, G, H), T3-treated (A, B), denervated (D, E, I) and denervated/T3-treated (F) regenerating muscle at 20 (A–F) and 30 (G–I) days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). B, C, E, F, H, I are darkfield photomicrographs. A, D, G are brightfield photomicrographs. Scale bar ⫽ 200 m produces a loss of MRF4 mRNA in uninjured muscle of adult Xenopus. This continuous down-regulation of MRF4 gene expression by denervation raises the pos- sibility that MRF4 expression may be induced by innervation and hence may be involved in mediating transcriptional responses to innervation. During Xeno- CONTROL OF EXPRESSION OF MYOGENIC REGULATORY FACTOR GENES pus myotome development, MRF4 mRNA is detected later, after MyoD and Myf-5 transcripts (Jennings, 1992), and its expression overlaps with the formation of neuromuscular connections (Blackshaw and Warner, 1976; Kullberg et al., 1977). It is interesting to note that in vitro studies have recently shown that MRF4 and the AchR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle of mammals, were found to be coexpressed at the formation of multinucleated spontaneously contracting myotubes (Rohwedel et al., 1998). This result and the fact that MRF4 is the MRF which preferentially transactivates the epsilon-promoter (Sunyer et Merlie, 1993) reinforce the possibility of an involvement of MRF4 in the regulation of nerve-regulated genes. The fact that the myogenin gene is up-regulated by denervation suggests that myogenin expression may compensate for the down-regulation of the MRF4 gene. It has been also shown that homozygous null mutant mice for MRF4 (⫺/⫺) exhibit fairly normal muscle and display an approximately threefold increase in myogenin expression, leaving open the possibility that myogenin also compensates for the loss of MRF4 in these embryos (Zhang et al., 1995). Nevertheless, it seems clear, in mammals, that myogenin and MRF4 do not have totally redundant functions, since myogenin (⫺/⫺) mice form myoblasts that fail to form myotubes efficiently in vivo. In Xenopus, this is not clear but our previous data (Nicolas et al., 1998a and b) point to the same conclusions. Whereas denervation does not have a consistent effect on MyoD expression in regenerating muscle of Xenopus, as observed in Xenopus adult muscles (Jennings, 1992), the fact that Myf-5 mRNA is transiently down-regulated by denervation in the first stage of regeneration is a surprising result. Indeed, previous reports indicated that no change or only little change occurred in the level of Myf-5 mRNA in denervated muscle of mice (Duclert et al., 1991) and chicken (Saitoh et al., 1993). CONCLUSION The cause of the discrepancy between Xenopus and mammalian or chicken results concerning MRF expression may reflect a diversity in response pathways to denervation or thyroid hormone treatment. In Xenopus, specific mechanisms may be responsible for physiological properties that are specific to Xenopus muscle. In mammals, muscle denervation has a dramatic effect on muscle morphology. In particular, denervated muscles are characterized by an important atrophy. In contrast, in Xenopus, denervated muscles did not present a different histological pattern as compared to innervated muscles (Jennings, 1992): atrophy of denervated muscle that is observed in mammals, was never detected in Xenopus. During regeneration of Xenopus muscle, Myf-5 and/or MyoD, which are expressed in activated satellite cells (Nicolas et al., 1996, 1998b) could be involved in the early events of the regeneration process. We can hypothesize that the decrease in Myf-5 mRNA levels 119 could be compensated by a stable and unaffected MyoD expression following variation in hormone levels and/or denervation. Thus, a stable MyoD expression could contribute to the initial differentiation events and play a crucial role in initiating the regeneration process as reported for mice (Megeney et al., 1996). In a second step, the “differentiation factors,” myogenin and MRF4, may contribute to the terminal differentiation events with partially overlapping functions (see above). Interestingly, gene dosage compensation of “early” or “late” MRF transcripts seems to follow epigenetic changes. A decreased expression of the early transcript Myf-5 is compensated by a stable expression of MyoD. Similarly, a decreased expression of the late transcript MRF4 is compensated by an increased expression of the other late MRF transcript, myogenin. We can speculate that the induction of myogenin gene and possibly the continuous expression of MyoD transcripts following denervation contribute to maintain the specific physiological properties of Xenopus muscle. EXPERIMENTAL PROCEDURES Animals Adult Xenopus laevis were maintained at 22°C in tap water and fed once a week. Muscle Injury and Sampling Animals were anesthetized with tricaine methane sulfate (MS222), and pure cardiotoxin from Naja mossambica nigricollis venom (Latoxan, France) (10⫺5M in 0.9% NaCl) was injected into the right anterior brachial muscle of the forelimb (Nicolas et al., 1996). We ascertained that intramuscular injection of the solvent alone did not induce any deterioration of the muscles. Samples for histological studies were plunged in isopentane and frozen in liquid nitrogen. For RT-PCR studies, samples were placed in cryotubes and immediately frozen in liquid nitrogen. Before that, to make sure of the completeness of the degeneration/ regeneration process, all the samples were analyzed in transverse cryosections under the light microscope. Muscle Denervation Prior to cardiotoxin injury, the anterior brachial muscle of adult Xenopus was denerved as follows. A double proximal ligature and a double distal ligature with silk thread, separated by 2–3 mm, were done one the brachial nerve, which was then resected between these ligatures. Hyperthyroidism Just after the cardiotoxin injection, animals were treated with 3,5,3⬘-triiodothyronine (T3); water in the breeding tanks was supplemented with T3 (5. 10⫺8 M), and this medium was changed every day for two months. Thyroid Hormone Assays T3 serum concentrations were determined by a specific immunoassay (Immo Phase) modified from the 120 NICOLAS ET AL. protocol recommended by Corning Medical Laboratories; in particular, the T3 antibody was diluted 10-fold. Preparation and Prehybridization of Tissue Sections The procedure for fixing, embedding and sectioning tissues was as for mouse embryos and was essentially the same as described by Wilkinson et al. (1987) with some modifications (Ontell et al., 1993). Briefly, tissues were fixed in 4% paraformaldehyde in PBS, dehydrated and infiltrated with paraffin. Then 6 m thick, serial sections were mounted on TESPA-coated RNase-free glass slides. Sections were deparaffinized in xylene, rehydrated, digested with proteinase K, postfixed, treated with dithiothreitol/iodoacetamine/N-ethylmaleimide (to reduce non-specific 35S-binding; Zeller and Rogers, 1989), treated with triethanolamine/acetic anhydride, washed and dehydrated. Probe Preparation The following probes were used to generate antisense cRNAs: (a) XMyoD template is a 598 nt 3⬘ fragment (BamHIEcoRI; position 872-1469) of XMyoD2-24 (Hopwood et al., 1989) subcloned in pGEM 4Z (Promega Biotec, Madison, WI), cut with BamHI and transcribed with SP6 RNA polymerase. (b) Xmyogenin template is a 522 nt fragment corresponding to a DNA sequence subcloned into pGEM7Zf(⫹), plamid pCJMG2 (Jennings, 1992), linearized with SphI and transcribed using SP6 RNA polymerase. (c) pSP73-XMyf5-2 template (Hopwood et al., 1991), was linearized using PstI and transcribed using SP6 RNA polymerase. The RNA probe was a 534 nt 3⬘ fragment (PstI-EcoRI, position 601–1134). (d) XMRF4, pCJM 4.2 (Jennings, 1992), was linearized using BamHI and transcribed using T7 RNA polymerase. The RNA probe was a 3⬘ fragment (BamHI-XhoI, position 721-3⬘ terminus). cRNA probes were made by in vitro transcription in the presence of 50 Ci [35S]UTP at 1,200 Ci/mmol (NEN research product), according to the manufacturer’s instructions (Promega Biotec, Madison, WI). However, unlabeled UTP was omitted from the reaction medium in order to achieve synthesis of RNA probes with a specific activity of 109 cpm/g. Probes were hydrolyzed to an average of 100 nucleotides by limited alkaline hydrolysis, according to Cox et al. (1984), for efficient hybridization, and used at 50,000 cpm/l hybridization solution. Thirty l of hybridization solution was loaded per section. Hybridization and Washing Procedures High-stringency conditions for hybridization and post-hybridization were followed. Sections were hybridized overnight at 53°C with post-hybridization washing in 2⫻SSC, 50% formamide, 50 mM DTT at 65°C for 30 min. Autoradiography was carried out with Kodak NTB-2 track emulsion, developed in Kodak D19 developer and stained lightly with Giemsa. Evaluation of Hybridization Signal In order to compare the intensity of hybridization signal with a given cRNA probe over time, sections at each stage were hybridized with the same probe preparation, washed, dipped into emulsion, exposed and developed together. Changes in the level of hybridization signal with a given cRNA probe over time were evaluated by taking dark-field micrographs, with a constant light intensity and a constant time of exposure of the film to the light source. Histology For the histological results presented, transverse frozen sections, 8m thick, were used. They were stained with hematoxylin and eosin. RNA Extraction Total RNA was purified by the method of Auffray and Rougeon (1980) and was monitored for quality by agarose gel electrophoresis and ethidium bromide staining. RT-PCR This was performed in one single tube, according to Goblet and Whalen (1995) and Lin-Jones and Hauschka (1996). First-Strand cDNA Synthesis One microgram of total RNA was used for RT-PCR for all primer pairs. The RNA was denatured briefly with 25 ng of random primer (Gibco BRL) at 65°C for 5 min. Then 1X RT buffer (ATGC), 1 mM each dNTP, 10 U of RNasin (Promega) and 200 units AMV Reverse Transcriptase (ATGC) were added to a final reaction volume of 20 l. Reverse transcription reactions were incubated for 10 min at room temperature, 60 min at 37°C, and 5 min at 95°C. PCR Amplification The whole reverse transcription reaction was diluted to 100 l final volume with 1X PCR buffer (ATGC). Multiplex PCR reaction was performed with a MRFspecific set of primers, and with a EF-1␣-specific sets of primers (see below), and 2 units of Taq polymerase (ATGC). Samples were overlaid with paraffin oil (80 l) and amplified in a thermocycler (Appligène, France). The cycling parameters were as follows: the initial cycle consisted of a 95°C denaturation for 5 min, 1 min at a 55°C annealing temperature, and 1 min at a 72°C extension temperature. The remaining cycles were for 30 sec at 95°C, 1 min at 55°C and 1 min at 72°C, with the final cycle having a 10 min extension at 72°C. Due to the different abundance of EF-1␣ and MRF transcripts, we performed 9 PCR cycles with MyoD, MRF4 or myogenin primer pairs or 12 PCR cycles with CONTROL OF EXPRESSION OF MYOGENIC REGULATORY FACTOR GENES Myf-5 primer pairs, before adding the EF-1␣ primers and continuing amplification for 19 additional cycles. One-fifth of the PCR sample was electrophoresed on 6% polyacrylamide gels. DNA was transferred onto Hybond-C super membrane (Amersham). Southern blots were hybridized with MRF and EF-1␣ radioactive probes. The amount of EF-1␣ PCR product was quantified using a Bio-imaging analyzer and NIH image analyzer software. This calibration allowed us to adjust the volumes of the PCR reactions loaded on the polyacrylamide gel. PCR Primers XMyf-5: 5⬘-ACTACTACAGTCTCCCAGGACAGAG-3⬘ (F); 5⬘-AGAGTCTGGAATAGGGAGGGAGCAT-3⬘ (R) (positions 524 –774, Hopwood et al., 1991). This primer pair produced a fragment of 250 bp from cDNA. XMyoD : 5⬘-AACTGCTCCGATGGCATGATGGATTA-3⬘ (F); 5⬘-ATTGCTGGGAGAAGGGATGGTGATTA-3⬘ (R) (positions 662–952, Hopwood et al., 1989). This primer pair produced a fragment of 289 bp from cDNA. XMRF4 : 5⬘-CTTTTACCTGGATGGAG-3⬘ (F); 5⬘-TGGTGGAGCTAAGACAT-3⬘ (R)(positions 147–309; Jennings, 1992). This primer pair produced a fragment of 162 bp from cDNA. Xmyogenin: 5⬘-CCAGCCCTTATTTCTTTTCAGACCA-3⬘ (F); 5⬘-AATCCCTGAGCCCTGTAATAAAACC-3⬘ (R) (positions 35–183; Jennings, 1992). This primer pair produced a fragment of 147 bp from cDNA. EF-1␣: 5⬘-CCTGAATCACCCAGGCCAGATTGGTG-3⬘ (F); 5⬘-GAGGGTAGTCTGAGAAGCTCTCCACG-3⬘ (R) (positions 1088 –1311; Krieg et al., 1989). This primer pair produced a fragment of 222 bp from cDNA. Calibration of the RT-PCR Assay In the first series of experiments, we used only one primer pair per PCR and 1 g of total RNA extracted from regenerating muscles 15 days P-I. The total number of cycles varied from 5 to 40 and the exponential step of the PCR reaction was determined. In a second set of experiments we determined the maximal RNA input by adding to the RT reaction serial dilutions of total RNA extracted from regenerating muscles 15 days P-I. The PCR was performed with subsaturating cycle number. In the latter experiments we determined the competitivity of the two primer pairs during the multiplex PCRs with 1 g of total RNA extracted from regenerating muscles 15 days P-I and a subsaturating number of cycles. The rate of signal intensity obtained during multiplex PCR was the same as the intensity proportion of separated PCR signals. Negative Controls Negative controls were performed with samples in which the reverse transcriptase or RNA or one of the primers was omitted, to detect eventual DNA contamination. All these controls remained consistently negative. 121 RNAse Protection Assay RNAse protections were performed using the following probes: Xenopus AChR ␣ subunit template is an EcoRI-BglII fragment corresponding to positions 237– 496 of the full-length SP65-AChR ␣ cDNA (Baldwin et al., 1988). EF-1␣, G1EF.BS subunit template is a PstIBstEII fragment corresponding to positions 790 – 879 of the full-length EF-1␣ cDNA (Krieg et al, 1989). ACKNOWLEDGMENTS We thank Drs J.B. 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